SEPARATION AND CHARACTERIZATION OF GRASSHOPPER HEMOLYMPH PHENOLOXIDASES BY SODIUM DODECYL-SULFATE POLYACRYLAMIDE-GEL ELECTROPHORESIS

1. A technique is described for the simultaneous localization, quantification and determination of the molecular weight of phenoloxidase after separating sample proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sensitivity of this method is 2.5 times that of the conventional spectrophotometric assay. We applied this technique to study the phenoloxidase from the hemolymph of 8 species of grasshoppers representing the subfamilies Melanoplinae and Oedipodinae and from adults of the cockroach, Periplaneta american and larvae of the wax moth, Galleria mellonella. 2. The molecular weights of phenoloxidases from various species of field-collected grasshoppers ranged from 25 kDa to 212 kDa, whilst that of the laboratory-reared Melanoplus sanguinipes was 184.6 kDa. The molecular weight of phenoloxidase from P. americana and G. mellonella was also 184.6 kDa. 3. Generally, the melanopline grasshoppers had one phenoloxidase species, while in the oedipodinid grasshoppers the number of phenoloxidases varied. 4. Addition of chymotrypsin to female M. sanguinipes hemolymph resulted in the formation of 2 phenoloxidase bands of activity after a 30 min incubation, indicating hydrolysis and activation of the phenoloxidase. 5. Intraspecies differences in number and molecular weights of phenoloxidase species were also observed in the field-collected grasshoppers. We speculate that in addition to isozymes, age and environmental factors may generate these differences in phenoloxidase patterns.